Monographs on 18 Theoretical and Applied Genetics

Edited by R. Frankel (Coordinating Editor), Bet-Dagan M. Grossman, Urbana· H. F. Linskens, Nijmegen P. Maliga, Piscataway· R. Riley, London Monographs on Theoretical and Applied Genetics

Volume 1 Meiotic Configurations A Source of Information for Estimating Genetic Parameters By 1. Sybenga (1975) Volume 2 Pollination Mechanisms, Reproduction and Plant Breeding By R. Frankel and E. Galun (1977) Volume 3 Incompatibility in Angiosperms By D. de Nettancourt (1977) Volume 4 Gene Interactions in Development By L.1. Korochkin (1981) Volume 5 The Molecular Theory of Radiation Biology By K.H. Chadwick and H.P. Leenhouts (1981) Volume 6 Heterosis Reappraisal of Theory and Practice Editor: R. Frankel (1983) Volume 7 Induced Mutations in Plant Breeding By W. Gottschalk and G. Wolff (1983) Volume 8 Protoplast Fusion Genetic Engineering in Higher Plants By Y.Y. Gleba and K.M. Sytnik (1984) Volume 9 Petunia Editor: K.C. Sink (1984) Volume 10 Male Sterility in Higher Plants By M.L.H. Kaul (1988) Volume 11 Tree Breeding: Principles and Strategies By G. Namkoong, H.C. Kang, and 1.S. Brouard (1988) Volume 12 The Wheat Rusts - Breeding for Resistance By D.R. Knott (1989) Volume 13 Population Dynamics and the Tribolism Model Genetics and Demography By R.F. Costantino and R.A. Desharnais (1991) Volume 14 Genetic Improvement of Tomato Editor: G. Kalloo (1991) Volume 15 Evolution and Function of Heterostyly Editor: S.C.H. Barrett (1992) Volume 16 Distant Hybridization of Crop Plants Editors: G. Kalloo and 1.B. Chowdhury (1992) Volume 17 in Plant Breeding By J. Sybenga (1992) Volume 18 Cytogenetics of the Festuca-Lolium Complex By P.P. lauhar (1993) p. P. lauhar Cytogenetics of the Festuca-Lolium Complex Relevance to Breeding

With 53 Figures

Springer-Verlag Berlin Heidelberg New York London Paris Tokyo Hong Kong Barcelona Budapest Prof. Dr. Prem P. lauhar USDA-ARS, Red River Valley Agricultural Research Center Northern Crop Science Laboratory State University Station Fargo, North Dakota 58105-5677 U.S.A.

Library of Congress Cataloging-in-Publication Data. lauhar, Prem P. Cytogenetics of the Festuca-Lolium complex: relevance to breeding/P.P. lauhar. p. cm.-(Monographs on theoretical and applied genetics: 18) Includes bibliographical references and index. ISBN-13: 978-3-642-84088-3 e-ISBN-t3: 978-3-642-84086-9 DO I: to. t 007/ 978-3-642-84086-9 1. Fescue-Breeding. 2. Lolium-Breeding. 3. Fescue-Cytogenetics. 4. Lolium-Cytogenetics. I. Title. II. Series. SB201.F48J38 1992 633.2'37-dc20 92-15946

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1993 Softcover reprint of the hardcover lst edition 1993 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting: Best-set Typesetter Ltd, Hong Kong 3113145-5 4 3 2 1 0 - Printed on acid-free paper To the memory of my father, whose life was a constant struggle for social justice and rights for all. Preface Considering the widespread occurrence and ecological diversity of grasses, their dominance over vast regions of our globe, and their prime importance to humankind, we, the experts, may congratulate ourselves on having become authorities on the most important single family of organisms in the world of life, rivaled only by the human family itself. G. Ledyard Stebbins 1987

From times immemorial man has relied heavily on grasses, directly and indirectly, for most of his basic needs. Grasses have had a profound impact on the development of human societies and influenced civilization - perhaps in more ways than any other crop. A paramount factor in the evolution of civilizations is a steady supply of food. Grasses were indeed essential to the "evolution" of wild and domestic grazing animals, which, in turn, also catalyzed the "evolution" of humankind. Thus, grasses and human civiliza• tion were inseparably associated, the association marked by intimate coevolution of grasses, herbivores, and man. Even today, on a world-wide basis, acreage under grasslands is es• timated to be twice that of cropland. However, grasslands are still one of the largest underdeveloped resources in the world. Knowledge acquired from an array of scientific disciplines should help tap their enormous potential for increasing agricultural productivity. It should also help preserve the vast global germplasm resources, the rich reservoirs of desirable genes. Festuca and Lalium are valuable genera of temperate grasslands. The two genera are closely related. They contain some very productive, nutritious, and well-adapted grasses widely used for agricultural and recreational purposes. They are an important source of feed for the livestock industry that provides us with wholesome meat, milk, wool, etc. They also play an important role in conserving and stabilizing soils. These grasses are widely distributed in temperate and cool climates in Europe, North Africa, South America, Asia, South and East Africa, Australia, and New Zealand; and they have been spread by man to several other parts of the world. Because of their importance in temperate agriculture, the Festuca• Lalium group of grasses have been extensively studied by agronomists, animal scientists, plant breeders, taxonomists, and cytogeneticists. However, no integrated information has been published on them. The results of various studies remain scattered in numerous journals and periodicals in different languages, some of which are not easily accessible to a common researcher. I have attempted, therefore, to compile and synthesize import• ant information on certain aspects of these grasses into this volume. The ultimate objective would be to use information from different fields of enquiry to improve the quantity and quality of forage and to stop or reduce VIII Preface genetic erosion. This book is, however, not intended to be an encyclopedic review of different topics. This book deals mainly with the cytogenetics of agronomically important grasses of the Festuca-Lolium complex and discusses their basic importance and breeding relevance. The perennial nature of these grasses makes them particularly amenable to cytogenetic and breeding manipulation. Modern biotechnological developments also hold considerable promise for their improvement. While the current available knowledge on the Festuca-Lolium group will be synthesized and discussed, what we do not know about them will also be highlighted. The book consists of 15 chapters, which deal with various facets of cytogenetics of the Festuca-Lolium group in relation to breeding. An appropriate introduction to each chapter and to several sections in different chapters puts the information in proper perspective to help the reader. Each chapter can be read independently, an arrangement that has inevitably led to some duplication between chapters. However, repetition has been kept to a minimum by giving cross-references. Some speculations have been made to stir ideas and interest among researchers. After all, science can and does build on speculates. Chapter 1 describes the general importance of the Festuca-Lolium group of grasses, their complementary attributes, and the promise they hold for agricultural and recreational purposes. Chapter 2 deals with various taxonomic treatments given to this fascinating group. Certain taxonomic controversies are highlighted and the multidisciplinary approach to revealing phylogeny emphasized. Karyotypic features in relation to evolution and divergence are discussed in Chapter 3. How species divergence in diploids is accompanied by massive changes in the amount of nuclear DNA is described. Discussion of genetic control of diploid-like meiosis in polyploid species of Festuca forms the main body of Chapter 4, the cyto• genetic basis of among ecotypes of hexaploid tall fescue is described in Chapter 5, and the phylogenetic and breeding impli• cations of these phenomena are discussed in Chapter 6, where possibilities of manipulating the diploidizing mechanism to effect intergeneric gene transfers are outlined. Chapter 7 deals with B chromosomes and their controversial role in stabilizing meiosis. and its value in plant improvement are described in Chapter 8, while Chapter 9 outlines the natural occurrence and artificial induction of haploidy in species of Lolium and Festuca, particularly F. arundinacea. Pairing in haploids shows the haplo-insufficiency of the genetic control of pairing; production of homo• zygous inbred lines is also briefly described. In Chapter 10, aneuploids are described and their use in elucidating cytogenetic architecture of hexaploid tall fescue is briefly stated. While Chapters 11 and 12 deal with intra- and inter-specific, and intergeneric hybridizations, genome relationships, and the development of superior cultivars, Chapter 13 discusses genomic balance in relation to fertility and its bearing on plant improvement. Preface IX

Chapter 14 highlights some of the biotechnological tools and their potential use in the genetic enrichment of different grasses. Chapter 15 concludes the book, discussing the past accomplishments and future outlook. The book is intended for workers engaged in the genetic improvement of the Festuca-Lolium group of grasses. Knowledge gained from these grasses may be relevant or applicable to other cross-pollinated herbage crops. I hope that this volume will provide useful information to geneticists and cytogeneticists, taxonomists and biosystematists, and agronomists and plant breeders. Students of cytogenetics and evolution should find this book particularly useful. A spectrum of other workers interested in general biology and agriculture will also find this volume a worthwhile reference. I am grateful to Professor H.F. Linskens for inviting me to write this book. Several scientists have read different chapters and given helpful suggestions. I am thankful to Dr. Elizabeth Kellogg (Harvard University), Dr. Richard Pohl (Iowa State University), Professor H. James Price (Texas A&M University), Dr. Craig W. Greene (College of the Atlantic, Bar Harbor, Maine), Dr. Mary Barkworth (Utah State University, Logan, Utah), Dr. Leila Shultz (Intermountain Herbarium, Logan, Utah), Professor Steven Aust (Biotechnology Center, Utah State University, Logan, Utah), Professor I.K. Vasil (University of Florida, Gainesville) and Professor B.V. Conger (University of Tennessee, Knoxville) for commenting on different chapters. Although several scientists have gone through various chapters, I take full responsibility for any errors, omissions, or misinterpretations. I am grateful to several scientists who provided photographs for this book: To Dr. Z. Bulinska-Radomska for Fig. 2.6; to Dr. V. Carnide for Figs. 3.2 and 3.3; to Dr. Georgia C. Eizenga and Ms. Etta M. Thacker for Figs. 9.1 and 9.2; to Dr. Robert C. Buckner for Figs. 1l.8b,c; to Mr. Hu Martin Thomas for Fig. 12.6; and to Ms. Sue Dalton for Figs. 14.3 to 14.6. My own family members have contributed significantly to this chal• lenging assignment in several ways. I am particularly grateful to my wife, Raj, who patiently waited for long hours in the night when I was trying to complete this book assignment. She is a constant source of encouragement. But for her understanding, I could not have accomplished whatever little I have done in science. Our daughter, Suneeta, did not see much of me when she was growing up and I thank her for her sacrifice. Once she commented; "I wish the day had thirty hours so that my dad could spend a few hours at home." Our sons, Rajiv and Sandeep, were a constant source of inspiration and encouragement. Last, but not least, my mother - an uneducated scientist - never failed to provide moral encouragement from her room in a nursing home.

Prem P. lauhar Contents

Chapter 1 The Festuca-Lolium Complex: a Fascinating Group of Grasses

1.1 Agronomic Importance ...... 2 1.1.1 Association Between Fungal Infection and Agronomic Traits ...... 3 1.2 Distribution ...... 3 1.3 Genetic Systems and Strategy for Germplasm Collection and Preservation ...... 5 1.3.1 Level of Hetero- or Homozygosity and Sampling ...... 6 1.3.2 Preservation of Germplasm ...... 6

Chapter 2 Taxonomic Treatments

2.1 General Considerations...... 9 2.2 Taxonomic Value of Epidermal Patterns and Leaf Sections ...... 10 2.3 Limitations Imposed by Natural ...... 11 2.4 The Lolium-Festuca-Vulpia Complex ...... 12 2.5 Lolium L...... 13 2.5.1 Salient Generic Features...... 14 2.5.2 The Outbreeding and Inbreeding Species...... 14 2.5.3 Crosses Between Outbreeders and Inbreeders: Possible Evolutionary Trends...... 15 2.5.4 Two Important Ryegrasses ...... 16 2.5.5 Systematic Mutations and Their Taxonomic Implications. . . 17 2.5.6 Vegetative Proliferation in Inflorescences: a Reproductive and Taxonomic Anomaly...... 17 2.6 Festuca L...... 19 2.6.1 Salient Generic Features...... 19 2.6.2 Classification into Sections ...... 20 2.7 Festuca arundinacea, a Compilospecies...... 23 2.8 Cytotaxonomic Affinities: Phylogenetic Considerations in the Festuca-Vulpia-Lolium Complex...... 24 XII Contents

Chapter 3 Karyotypes and Species Evolution and Divergence

3.1 Karyotypic Features and Taxonomy ...... 29 3.2 General Techniques of Studying Somatic Chromosomes . . .. 30 3.2.1 Conventional Staining ...... 30 3.2.2 General Karyotypic Features of Lolium Species...... 31 3.2.3 General Karyotypic Features of Festuca Species...... 31 3.2.4 Giemsa Staining ...... 32 3.2.4.1 Lolium L...... 32 3.2.4.2 Festuca L...... 33 3.3 Chromosome Size Variation Among Diploids and Polyploids ...... 33 3.4 Evolution by Increase in Chromosome Numbers ...... 34 3.5 Evolution by Changes in DNA Content ...... 34 3.5.1 Lolium L...... 34 3.5.2 Festuca L...... 36 3.6 DNA Content and Growth Habit...... 37 3.7 Constitutive Heterochromatin ...... 38 3.8 Nucleolar Organizer Activity ...... 38

Chapter 4 Meiosis in Diploid and Polyploid Species

4.1 General Technique of Studying Meiosis ...... 43 4.2 Meiosis in Diploids...... 43 4.3 Diploid-Like Meiosis in Polyploids ...... 43 4.3.1 Genetic Control of Diploid-Like Meiosis and Disomic Inheritance in Hexaploid Tall Fescue...... 44 4.3.1.1 Meiotic Analysis of 10 x 10 Dialle\: Chromosome Pairing in a Critical Monosomic ...... 46 4.3.1.2 Disomic Inheritance...... 48 4.3.1.3 The Haplo-Insufficiency Hypothesis ...... 48 4.3.2 Occurrence of Haplo-Insufficient Diploidizing Genes in Other Polyploids...... 49 4.3.3 Possible Dosage Effect of Diploidizing Gene(s) ...... 50 4.3.4 Can Tall Fescue Be Considered Autoallohexaploid? ...... 51 4.3.5 Probable Location of the Diploidizing Gene(s) in Tall Fescue ...... 51 4.4 Breakdown of Diploidizing Mechanism in Haploid Complements in Hybrids...... 52 4.4.1 Haplo-Insufficiency of Diploidizing Gene(s) ...... 52 4.4.2 Possible Effects of Parental Genotypes...... 53 4.5 Chromosome Pairing in Polyhaploids ...... 54 4.6 B Chromosomes in Relation to Diploidization of Tall Fescue ...... 54 Contents XIII

4.7 Different Diploidizing Mechanisms Compared and Contrasted ...... 55

Chapter 5 Reproductive Isolation Barriers Within the Tall Fescue Complex

5.1 Cytogenetic Basis of Reproductive Isolation...... 57 5.1.1 Regular Meiosis ...... 57 5.1.2 Irregular Meiosis with Multivalent Formation...... 59 5.1.3 Irregular Meiosis with Multivalents and Univalents ...... 59 5.1.4 Irregular Meiosis with Univalents ...... 62 5.2 Various Other Meiotic Abnormalities in Hybrids Between Diverse Ecotypes ...... 62 5.3 Occurrence of Univalents at Metaphase I: a Case of Desynapsis ...... 63 5.4 Formation of Multivalents: Genetic Bases ...... 64 5.5 Haplo-Insufficiency of the Pairing Control Genes? ...... 67 5.6 Genetic Repression of Diploidizing Gene(s): an Example. . . 68

Chapter 6 Phylogenetic and Breeding Implications of the Genetic Control of Chromosome Pairing

6.1 Phylogenetic Relationships ...... 71 6.2 Evolutionary Implications...... 72 6.2.1 Introgression of Characters...... 72 6.2.2 Changes in Basic Chromosome Numbers...... 72 6.3 Breeding Implications ...... 73 6.3.1 Estimation of Inbreeding in Tall Fescue...... 73 6.3.2 The Concept of "Chromosome Combining Ability" and Formulation of Breeding Programs ...... 74 6.3.3 Use of Genetic Bridges ...... 74 6.3.4 Breeding Dodecaploid Tall Fescue and Derivatives ...... 75 6.3.5 Interspecific Gene Transfers ...... , ...... , 75

Chapter 7 B Chromosomes

7.1 Occurrence and Adaptive Value ...... 77 7.1.1 General Occurrence ...... 77 7.1.2 B Chromosomes in Festuca pratensis Huds.: Adaptive Significance? ...... , 78 7.1.3 B Chromosomes in Latium perenne L...... 80 7.2 Pairing Among B Chromosomes in Hexaploid Tall Fescue . .. 80 7.3 Effect on Genetic Recombination...... 83 7.4 Suppression of Homoeologous Pairing in Hybrids...... 83 7.5 B Chromosomes in Relation to Plant Breeding ...... , 85 XIV Contents

Chapter 8 Natural and Induced Polyploidy

8.1 Natural Polyploidy and Its Adaptive Value in Festuca ...... 87 8.2 Absence of Natural Polyploidy in Lalium...... 88 8.3 Induced Polyploidy in Lalium ...... 89 8.3.1 Colchicine Treatment...... 89 8.3.2 Meiotic Doubling...... 89 8.4 Distinguishing Features of Synthetic Polyploids of Lalium ... 90 8.4.1 Morphological Features...... 90 8.4.2 Isozyme Patterns...... 91 8.4.3 Nucleolar Organizing Regions ...... 91 8.4.4 Meiosis and Fertility...... 91 8.4.5 Ribulose 1,5-Bisphosphate Carboxylase Activity ...... 92 8.4.6 Colchicine-Induced Variation...... 93 8.5 Induced Polyploidy in Festuca ...... 93 8.5.1 Tetraploid Meadow Fescue...... 94 8.5.2 Dodecaploid Tall Fescue...... 94 8.5.3 Synthesis of Triploid Hybrids Between F. pratensis and F. apennina [= F. pratensis var. apennina] ...... 96 8.6 Occurrence of Aneuploids in Synthetic Polyploids ...... 99 8.6.1 Sources of Aneuploidy...... 99 8.6.2 Aneuploid Frequency...... 99 8.6.3 Undesirable Effects of Aneuploidy...... 100 8.7 Polyploidy in Relation to Plant Breeding ...... 101 8.7.1 Importance of Cytological Diploidization in Improvement of Synthetic Polyploids...... 102 8.7.2 Polyploid Material of Breeding Value ...... 103

Chapter·9 Haploidy

9.1 Lalium Haploids ...... 107 9.1.1 Anther Culture ...... 107 9.1.2 Wide Hybridization ...... 108 9.2 Festuca Haploids...... 109 9.2.1 Polyhaploids of Hexaploid Tall Fescue...... 109 9.2.2 Doubled Haploids of Tall Fescue ...... 110 9.2.3 Chromosome Pairing in Polyhaploids ...... 110 9.2.4 Polyhaploids of Decaploid Tall Fescue...... 112

Chapter 10 Aneuploids

10.1 Lalium ...... 113 10.1.1 Isolation of Trisomies...... 113 10.1.2 Meiosis in Trisomies...... 115 Contents xv

10.1.3 Location of Genes ...... 115 10.2 Hexaploid Tall Fescue...... 116 10.2.1 Isolation of Monosomics and a Nullisomic...... 116 10.2.2 Trisomics ...... 117 10.2.3 Future Work...... 118

Chapter 11 Intra- and Inter-Specific Hybridization, Genome Relationships, and Plant Improvement

11.1 General Importance...... 121 11.2 Crossing Techniques...... 122 11.2.1 Choice of Parents ...... 122 11.2.2 Hybridization...... 122 11.3 The Embryo Rescue Technique...... 122 11.4 Extensive Hybridization Work...... 124 11.5 General Meiotic Abnormalities in Wide Hybrids ...... 125 11.6 Phylogenetic Proximity Among Species of the Lalium-Festuca Complex: a Generalized Picture. . . .. 125 11.7 Intraspecific Hybrids in Lalium L...... 127 11.7.1 Cytoplasmic Male Sterility ...... 127 11.7.2 Diploid x Tetraploid Crosses...... 127 11.8 Intraspecific Hybrids in Festuca L...... 128 11.8.1 Inter-Ecotype Hybrids...... 128 11.8.2 Hybrids Between Material at Different Levels ...... 128 11.9 Interspecific Hybrids in Lalium ...... 129 11.9.1 General Genome Relationships ...... 129 11.9.1.1 Intragroup and Intergroup Crosses ...... 130 11.9.1.2 Do Lalium Species Have the Same Basic Genome? ...... 132 11.10 Hybrids Between the Two Ryegrasses: Development of Superior Germplasm ...... 132 11.10.1 Diploid Hybrids ...... 132 11.10.2 Tetraploid Hybrids...... 133 11.10.3 Preferential Pairing Between Lp-Lp and Lm-Lm Chromosomes ...... 137 11.10.4 Cytological Diploidization of Tetraploid Hybrids ...... 137 11.10.5 Agronomically Useful LmLm LpLp Hybrids...... 138 11.10.6 Recombination of Traits via a Triploid Hybrid ...... 138 11.11 Interspecific Hybrids in Festuca ...... 139 11.11.1 F. pratensis (2x) x F. arundinacea (6x) ...... 139 11.11.2 F. pratensis (2x) x F. glaucescens (4x) ...... 140 11.11.3 F. pratensis (2x) x F. mairei (4x) ...... 140 11.11.4 F. pratensis (2x) x F. apennina De Not. (4x) ...... 141 11.11.5 F. arundinacea (6x, 8x, lOx) x F. mairei (4x) ...... 141 11.11.6 F. glaucescens (4x) x F. mairei (4x)...... 142 XVI Contents

11.11.7 Chromosome Pairing Relationships Among Diploid Species of Festuca ...... 142 11.11.8 General Conclusions on Genome Relationships Among Species of Festuca ...... 144 11.12 F. arundinacea (6x) x F. gigantea (6x) Hybridization: Development of Superior Germplasm...... 144 11.12.1 Chromosome Relationships ...... 144 11.12.2 Material of Breeding Value...... 147

Chapter 12 Intergeneric Hybridization, Genome Relationships, and Plant Improvement

12.1 Possible Affinity Between Festuca and Other Grass Genera 149 12.2 General Affinities Between Lolium and Festuca ...... 149 12.3 Natural Hybrids ...... 150 12.4 Synthetic Hybrids...... 151 12.4.1 Lolium perenne (2x) or L. multiflorum (2x) x F. pratensis (2x) ...... 151 12.4.2 Lolium multiflorum (2x) or L. perenne (2x) x F. arundinacea (6x) ...... 155 12.4.2.1 F) Hybrids...... 155 12.4.2.2 Amphiploids ...... 159 12.4.3 Lolium perenne (2x) x F. rubra (6x) ...... 160 12.4.4 Lolium multiflorum (2x) x F. arundinacea var. letourneuxiana (lOx) ...... 161 12.4.5 Lolium perenne (2x) or L. multiflorum (2x) x F. gigantea (6x) ...... 161 12.4.5.1 Tetraploid F) Hybrids ...... 161 12.4.5.2 Amphidiploids...... 161 12.4.5.3 Pentaploid F) Hybrids ...... 162 12.5 Variation in Chromosome Size Between Diploid Species and Pairing in Their Hybrids...... 162 12.5.1 Diploid F) Hybrids...... 162 12.5.2 Autoallotriploid and Trispecific Hybrids...... 164 12.5.3 Amphidiploids...... 165 12.6 Intergeneric Hybridization and Production of Superior Breeding Material ...... 165 12.6.1 Genome Relationships Among Three Important Diploid Species...... 165 12.6.2 Superior (L. multiflorum or L. perenne x F. pratensis) Amphidiploids...... 171 12.6.3 Controlled Transfer of F. arundinacea Genes into L. multiflorum ...... 172 12.6.4 Superior Cultivars from L. multiflorum x F. arundinacea Hybridization...... 173 Contents XVII

12.6.4.1 Kenhy, an "Alien Substitution" Line ...... 174 12.6.4.2 Low-Perloline Derivatives...... 174 12.6.4.3 Identification of Alien "Blood" in Hybrid Derivatives...... 176

Chapter 13 Genomic Balance in Relation to Hybrid Fertility and Plant Improvement

13.1 Width of the Cross and Crossing Success ...... 177 13.2 Choice of Parents ...... 178 13.3 Choice of Female Parent...... 179 13.4 Genomic Constitution and Hybrid Fertility ...... 179 13.4.1 Hybrids Involving the Three Important Diploid Species .... 179 13.4.2 Use of "Interspecific" Hybrids as Parents...... 181 13.4.3 Hybrids Involving Diploid and Polyploid Species ...... 181

Chapter 14 Biotechnology in Festuca-Lolium Improvement

14.1 General Scope and Importance: Genetic Engineering ...... 183 14.2 Meristem-Tip Culture: a Method to Eliminate Pathogens. .. 184 14.3 In Vitro Callus Cultures and Screening for Tall Fescue Endophyte...... 185 14.4 Organogenesis and Embryogenesis ...... 185 14.4.1 Organogenesis...... 186 14.4.2 Somatic Embryogenesis...... 186 14.5 In Vitro Cultures and Plant Regeneration ...... 186 14.5.1 Lalium ...... 187 14.5.2 Polyploid Species of Festuca ...... 187 14.6 Anther Culture ...... 189 14.7 Somaclonal and Gametoclonal Variation...... 190 14.8 Colchicine-Induced Variation ...... 192 14.9 Environmentally Induced Somatic Variation...... 192 14.10 Cytogenetic Manipulation of Hybrid Material...... 193 14.11 In Vitro Chromosome Doubling ...... 193 14.11.1 Meristem Cultures ...... 193 14.11.2 Callus Cultures...... 195 14.12 Protoplast Cultures: Regeneration of Plants ...... 195 14.13 Genetic Manipulation at the Cellular Level...... 199

Chapter 15 Conclusions and Perspectives 203

References ...... 209

Subject Index...... 243